1. Field of Invention
This invention relates generally to the field of spectroscopic detection, identification and/or characterization of substances and, more particularly, to fluorescence detection, identification and/or characterization performed by an integrated microanalytical system, as well as to materials, fabrication techniques and methods of use of such a microanalytical system.
2. Description of the Prior Art
Optical spectroscopy is a common technique for the detection, identification and/or characterization of many substances including atoms, molecules, ions, polymers, thin films, biological substances, among others. We describe herein primarily the detection of molecules pursuant to various embodiments of the present invention, understanding thereby that the descriptions herein are not limited simply to detection of molecules but can include identification, characterization and the acquisition of other information concerning the material under study. For economy of language, we use “detection” to refer to all such process. Similarly, the descriptions herein are not limited to molecules but can include other forms of matter as apparent to those having ordinary skills in the art, collectively described herein as “molecules”.
One important type of optical spectroscopy involves the excitation of a molecule to an excited electronic state, typically by absorption of radiation in the ultraviolet, visible or near-infrared regions of the spectrum. The electronically excited molecule then returns to its ground state, or to a lower-lying electronic state, accompanied by the emission of radiation. Detection and measurement of the properties of this emitted radiation provide important information concerning the molecule.
Many molecules of interest are not conveniently detected directly by fluorescence detection but advantageously employ a fluorescence-facilitating molecule selectively attached to the sample molecule whose detection and/or characterization is desired. That is, a molecule having favorable properties for fluorescence detection (typically a dye) is chosen or manufactured such that it selectively bonds to the sample whose detection is desired. The sample thus “tagged” is then subject to fluorescence detection. To be concrete in our descriptions, we focus chiefly on those examples in which tagging a sample with a dye molecule is an advantageous preliminary step in fluorescence detection. However, this is by way of illustration and not limitation since various embodiments of the present invention can be employed when several different tagging dyes are used, or in those cases in which the sample itself can be subject to fluorescence detection without the need for tagging. In addition, we use the term “dye” for economy of language to indicate any fluorescence-facilitating substance bonded to the sample, not restricted to other, possibly more restrictive, chemical definitions of dyes.
A distinction is sometimes made between “phosphorescence” and “fluorescence” with fluorescence indicating radiation emitted from the absorbing molecule essentially immediately following absorption and excitation; that is, emission follows absorption by less than about 1 millisecond. Phosphorescence may be used to denote a longer-delayed emission following absorption. However, such distinctions are not necessary in describing embodiments of the present invention. For economy of language, we use “fluorescence” to indicate emission of radiation following absorption, irrespective of the time delay between absorption and emission.
An important characteristic of fluorescence spectroscopy (or “fluorescence detection”) is its high sensitivity, often achieving detection limits several orders of magnitude lower than detection limits achievable with other analytical techniques. Thus, fluorescence detection is a widely used laboratory bench technique for bioassay applications as well as other analyses. However, bench-top systems are typically expensive as well as bulky, unsuited for use in the field or at remote locations.
Physicians, biologists and others routinely use bench-top instrumentation typically costing in excess of $100,000 to inspect biological fluids, excite reactions in chemicals to generate structural information, or to detect toxins by tagging molecules with fluorescing agents. These bench-top instruments typically include an enclosed laser that contributes to the physical size and expense. Miniaturization of this equipment would be expected to lead to a drastic reduction in cost as well as portability. Furthermore, since a miniaturized system is likely to be highly integrated, its use by laypersons is expected to be feasible. Thus, a need exists in the art for miniaturized fluorescence detection analytical systems including systems with lower cost, improved portability as well as high functionality and reliability.